Compositions and methods for immunotherapy

ABSTRACT

The present invention provides immunoresponsive cells, including T cells, cytotoxic T cells, regulatory T cells, and Natural Killer (NK) cells, expressing at least one of an antigen recognizing receptor and one of a chimeric costimulatory receptor. Methods of using the immunoresponsive cell include those for the treatment of neoplasia and other pathologies where an increase in an antigen-specific immune response is desired.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application SerialNo PCT/US2013/063097 filed Oct. 2, 2013, which claims priority to U.S.Provisional Patent Application Ser. No. 61/709,072 filed Oct. 2, 2012,the contents of each of which are incorporated by reference in theirentirety, and to each of which priority is claimed.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Apr. 1, 2015. Pursuant to 37 C.F.R.§1.52(e)(5), the Sequence Listing text file, identified as072734_(—)0147 USSEQ.txt, is 28,207 bytes and was created on Apr. 1,2015. The Sequence Listing, electronically filed herewith, does notextend beyond the scope of the specification and thus does not containnew matter.

BACKGROUND OF THE INVENTION

Prostate cancer is the most frequent cancer in males in the UnitedStates and the cause of nearly 31,000 deaths per year. When diagnosedearly, cancer can be effectively treated by surgery or radiation.Postsurgical residual disease requires radiation and/or hormonaltherapy, which may prevent tumor progression and metastasis. At present,there is no curative treatment for hormone refractory, metastaticprostate cancer. Immunotherapy is a targeted therapy that in principleprovides for the treatment of such cancers.

Targeted T ceil therapies utilizing genetically modified autologous Tcells are beginning to show evidence of therapeutic efficacy in melanomaand indolent B cell malignancies. Current T ceil engineering strategiesretarget patient T cells to tumor antigens through a transduced T cellreceptor (TCR) or a chimeric antigen receptor (CAR). The newfoundability to induce potent immune responses, however, commands the need toconfine immune attacks to the tumor and avoid reactions against normaltissues that may express the targeted antigen. Alas, the limitedavailability of truly tumor-restricted antigens often precludesachieving highly specific targeting is the limited availability of trulytumor-restricted antigens. Accordingly, new methods of treatingneoplasia are urgently required.

SUMMARY OF THE INVENTION

The present invention generally provides immunoresponsive cells,including T cells and Natural Killer (NK) cells, expressing an antigenbinding receptor (e.g., CAR or TCR) having immune cell activatingactivity and a chimeric co-stimulating receptor (CCR), and methods ofuse therefore for the treatment of neoplasia, infectious disease, andother pathologies.

In one aspect, the invention provides an isolated immunoresponsive cellhaving an antigen recognizing receptor that binds a first antigen withlow affinity, where the binding activates the immunoresponsive cell, anda chimeric co stimulating receptor (CCR) that binds a second antigen andstimulates the immunoresponsive cell.

In another aspect, the invention provides a method of inducing tumorcell death in a subject, the method comprising administering aneffective amount of an immunoresponsive cell comprising an antigenrecognizing receptor that binds a first antigen with low affinity, wherethe binding activates the immunoresponsive cell, and a chimericco-stimulating receptor (CCR) that binds a second antigen and stimulatesthe immunoresponsive cell, thereby inducing tumor cell death in thesubject.

In still another aspect, the invention provides a method of treating orpreventing a neoplasia in a subject, the method comprising administeringan effective amount of an immunoresponsive cell comprising an antigenrecognizing receptor that binds a first antigen with low affinity, wherethe binding activates the immunoresponsive cell, and a chimericco-stimulating receptor (CCR) that binds a second antigen and stimulatesthe immunoresponsive cell, thereby treating or preventing a neoplasia inthe subject.

In yet another aspect, the invention provides a method of treatingprostate cancer in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of a Tcell comprising an antigen recognizing receptor that binds PSCA or CD19with low affinity, where the binding activates the immunoresponsivecell, and a chimeric co-stimulating receptor (CCR) that binds PSMA andstimulates the immunoresponsive cell, thereby treating prostate cancerin the subject.

In still another, the invention provides a method for producing anantigen-specific immunoresponsive cell, the method involving introducinginto the immunoresponsive cell a nucleic acid sequence that encodes achimeric co-stimulating receptor (CCR), where the chimericco-stimulating receptor has an antigen-binding domain coupled to anintracellular signaling domain that stimulates an immunoresponsive cell,where the immunoresponsive cell has an antigen recognizing receptor thatbinds a first antigen with low affinity, wherein the binding activatesthe immunoresponsive cell.

In a related aspect, the invention provides a pharmaceutical compositioncomprising an effective amount of an immunoresponsive cell of theinvention (e.g., a tumor antigen-specific T cell in a pharmaceuticalcomposition for the treatment of neoplasia) in a pharmaceuticallyacceptable excipient.

In an additional aspect, the invention provides a kit for treatment of aneoplasia, pathogen infection, an autoimmune disorder, or an allogeneictransplant, the kit containing an immunoresponsive cell having anantigen recognizing receptor that binds a first antigen and activatesthe immunoresponsive cell, and a chimeric co-stimulating receptor (CCR)that binds a second viral antigen and stimulates the immunoresponsivecell. The kit may further comprise written instructions for using theimmunoresponsive cell for the treatment of a subject having a neoplasia,a pathogen infection, an autoimmune disorder, or an allogeneictransplant.

In various embodiments of any of the aspects delineated herein, theimmunoresponsive cell is selected as having an antigen recognizingreceptor with low affinity. This may involve selecting theimmunoresponsive cell as having an antigen recognizing receptor thatbinds a first antigen with low affinity. In various embodiments of anyof the aspects delineated herein, the antigen recognizing receptor isselected as having low affinity for expression in the cell. This mayinvolve introducing a second nucleic acid sequence that encodes achimeric antigen receptor, where the chimeric antigen receptor comprisesa second antigen-binding domain coupled to a second intracellularsignaling domain that activates an immunoresponsive cell. In variousembodiments of any of the aspects delineated herein, the antigenrecognizing receptor is a T cell receptor (TCR) or chimeric antigenreceptor (CAR). In various embodiments, the intracellular signalingdomain of said antigen recognizing receptor is the CD3-chain signalingdomain. In various embodiments, the intracellular signaling domain ofthe chimeric co-stimulating receptor (CCR) is a CD97, CD11a-CD18, CD2,ICOS, CD27, CD154, CD5, OX40, 4-1BB or CD28 signaling domain.

In various embodiments of any of the aspects delineated herein, theantigen recognizing receptor is exogenous or endogenous. In variousembodiments of any of the aspects delineated herein, the antigenrecognizing receptor is recombinantly expressed. In various embodiments,the antigen recognizing receptor is expressed from a vector. In variousembodiments, the chimeric co-stimulating receptor (CCR) is expressedfrom a vector. In particular embodiments, the immunoresponsive cellexpresses a recombinant or an endogenous antigen receptor that is 19z1or Pz1.

In various embodiments of any of the aspects delineated herein, theimmunoresponsive cell is a T cell, a Natural Killer (NK) cell, acytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonicstem cell, or a pluripotent stem cell from which lymphoid cells may bedifferentiated. In various embodiments of any of the aspects delineatedherein, the immunoresponsive cell of any one of claims 1-9, where saidimmunoresponsive cell is autologous.

In various embodiments of any of the aspects delineated herein, theantigen is a tumor or pathogen antigen. In various embodiments of any ofthe aspects delineated herein, one or more antigen-binding domains aretumor antigen-binding domains. In various embodiments of any of theaspects delineated herein, the antigens or tumor antigens are selectedfrom CAIX, CEA, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34,CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, a cytomegalovirus(CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP,Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT,IL13R-a2, x-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-AI,MUC1, Mesothelin, NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4),PSCA, PSMA, ROR1, TAG-72, VEGF-R2, and WT-1. In various embodiments, thefirst and second antigens are selected from CD133, a cytomegalovirus(CMV) infected cell antigen, erbB2, KDR Mesothelin, NKG2D ligands,NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, CD19, VEGF-R2, and WT-1.In particular embodiments, the first and second antigens are selectedfrom HER2, MUC1, CD44, CD49f, EpCAM, CEA, CD133, a cytomegalovirus (CMV)infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, KDR,Mesothelin, NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA,PSMA, VEGF-R2, or WT-1. In specific embodiments, the first and secondantigens are selected from CD10 and CD19. In other embodiments, thefirst and second antigens are selected from CD56 and CD138. In certainembodiments, the first and second antigens are selected from mesothelin,folate receptor-a, CD44, and CD133.

In various embodiments of any of the aspects delineated herein, theneoplasia is selected from the group consisting of prostate cancer,breast cancer, B cell leukemia, multiple myeloma, and ovarian cancer. Invarious embodiments of any of the aspects delineated herein, the methodreduces the number of tumor cells, reduces tumor size, and/or eradicatesthe tumor in the subject.

In various embodiments, the neoplasia is prostate cancer and the firstand second tumor antigens are distinct antigens selected from PSCA,PSMA, CD19, CD133, a cytomegalovirus (CMV) infected cell antigen,erb-B2, KDR Mesothelin, NKG2D ligands, NY-ES0-1, oncofetal antigen(h5T4), VEGF-R2, and WT-I. In various embodiments, the neoplasia isbreast cancer and the first and second tumor antigens are distinctantigens selected from HER2, MUC1, CD44, CD49f, EpCAM, CEA, CD133, acytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM,erb-B2,3,4, FBP, KDR, Mesothelin, NKG2D ligands, NY-ES0-1, oncofetalantigen (h5T4), PSCA, PSMA, VEGF-R2, or WT-1. In particular embodiments,the neoplasia is B cell leukemia and the first and second tumor antigensare selected from CDIO and CD19. In certain embodiments, the neoplasiais multiple myeloma and the first and second tumor antigens are selectedfrom CD56 and CD138. In various embodiments, the neoplasia is ovariancancer and the first and second tumor antigens are distinct antigensselected from mesothelin, folate receptor-a, CD44, and CD133.

The invention provides compositions and methods that provide for T celltargeting of tumor cells. Compositions and articles defined by theinvention were isolated or otherwise manufactured in connection with theexamples provided below. Other features and advantages of the inventionwill be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are graphics depicting chimeric antigen receptor (CAR) andchimeric costimulatory receptor (CCR) vector design and expression viatransduction of primary human T cells. (A) depicts generation of CARs byfusing heavy and light chains of immunoglobulin variable domains to theCD8 transmembrane domain, which is fused to the cytosolic signalingdomains of CD3. By using an Internal Ribosomal Entry Site (IRES) toenable bicistronic expression, CAR expression can be easily detected bycorrelation to dsRED fluorescence (data not shown). The CCR wasgenerated by fusing an scFv to a CD28 transmembrane and signalingdomain¹⁵, fused to a 4-1BB (aka CD137) cytosolic signaling domain.²¹ CCRexpression can be correlated to the bicistronic expression of hrGFP(data not shown). Abbreviations: LTR—Long Terminal Repeat; SD—SpliceDonor site; SA—Splice Acceptor site; VH or V_(L)—Variable Heavy or Lightdomains, respectively; EC—Extracellular domain; TM—Transmembrane domain;C—Cytosolic domain; IRES—Internal Ribosomal Entry Site; dsRED—Discosomasp. Red fluorescent protein, hrGFP—Human Recombinant Green FluorescentProtein (B) depicts representative transduction efficiencies of primaryhuman T cells using these retroviral vectors. (C) depicts transductionof CTLs with different and multiple CARs for the present studies.

FIGS. 2A-D show that dual-receptor, CAR/CCR-mediated activation of humanT cells allowed for robust CTL function, long-term proliferation, andenhanced tumor eradication upon binding of two antigens. (A) shows thatT cells expressing chimeric receptors lysed cells positive for antigenwhen the CAR specific to CD19 is expressed by T cells in CTL assays,compared to untransduced or P28BB transduced T cells. Plots arerepresentative of n>4 experiments, with error bars representing standarddeviation of the mean of 3 replicates. (B) shows long-term proliferationof T cells by absolute T cell counts over 31 days of T cells expressingnone, one, or both chimeric receptors that were co-cultured with humantumor cell lines expressing both or either antigen alone. Arrowsindicate re-stimulation of T cells using freshly irradiated tumor cells.Only when dual-receptor expressing T cells encounter both antigens isrobust long-term proliferation observed. Plots are representative of n>4experiments with error bars representing standard deviation of the meanof 3 replicates. (C) depicts the efficacy of systemic tumor eradicationby tumor-sensing T (TTS) cells assessed by infusing 1.0×10⁶ T cellsintravenously (IV) into NSG mice bearing luciferase expressingCD19⁺PSMA⁺ PC3 human prostate tumor. Tumor burden was quantitativelymeasured weekly by using BLI. Images of two representative mice fromeach group are shown with the pixel intensity from the luminescence oftumors represented in color. An average of tumor burden was plotted witherror bars representing standard deviation from the mean of values from6 mice per group. (D) depicts selective eradication of DP tumors using atri-tumor mouse model by subcutaneously injecting 1×10⁶ PC3 tumors cellseach of cells positive for CD19 alone into the left flanks, cellspositive for PSMA alone into the right flanks, and cells positive forboth CD19 and PSMA into the backs of the mice. T cells expressing either19z1, P28BB, or both 19z1+P28BB of the chimeric receptors were infusedintravenously 7 days post tumor infusion. Representative images of 2mice per group bearing these tumors are shown with luminescence oftumors represented in color. Tumors were quantitatively measured usingcalipers and tumor volumes were plotted versus time for each tumor.Error bars represent standard deviation from the mean of 6 mice.Statistical significance was determined using two-tailed unpaired ttests to compare values obtained from 19z1 T cells and 19z1+P28BB Tcells and p values are represented as * for <0.05 or ** for <0.01.

FIGS. 3A-E depict that tumor-sensing T_((TTS)) cells selectivelyeradicated human prostate tumors when targeting two prostate tumorantigens (A) depicts the evaluation of three different scFvs specific toPSCA for their assembly into bispecific antibodies that containspecificity for CD3 as well. T cells were co-cultured at ratio of 20:1with PSCA PC3 tumor cells and antibodies added at varying amounts andspecific lysis was measured. (B) depicts generation of CARs using theanti-PSCA scFvs that display varied efficacy in cytotoxicity assays. TheCAR mediated specific lysis of target cells expressing PSCA corroboratedthe reduced efficacy of the LzI scFv by requiring a 50 fold higheffector: target ratio to achieve the same level of lysis of that foreither HzI or MzI. (C) and (D) depict selective eradication of systemicprostate tumors expressing PSCA and PSMA was investigated by using theseinefficient scFvs. Tumors (FIG. 5) were established and treated asdescribed in FIG. 2. After 14 days, 1.0×10⁶ chimeric receptor positive Tcells for MzI+P28BB (FIG. 3C) or LzI+P28BB (FIG. 3D) were infusedintravenously. Images of two representative mice from each group areshown with luminescence from tumors represented in color (fromBlue=5×10⁵ to Red=2×10⁷ photons). The average tumor burden wasquantified by luminescence and plotted with error bars representingstandard deviation from the mean of values from 5 mice per group. Twomice that received PSMA tumor (green line) died after day 49 andtherefore the mean value for luminescence was averaged from 3 values fordays 56 and 63. FIG. 3E Selective antitumor responses to only PSCA⁺PSMA⁺tumors was achieved by LzI+P28BB T cells in mice that also hadPSCA⁺PSMA⁺ and PSCA⁺PSMA⁺ tumors, similar to FIG. 2D. Statisticalsignificance was determined using two-tailed unpaired t tests to comparevalues obtained from LzI T cells and LzI+P28BB T cells and p values arerepresented as * for <0.05 or ** for <0.01.

FIGS. 4A-D depict enhanced cytokine secretion and Bc1xL expression isfound by TTS cells when co-cultured on DP tumors. (A) depicts multiplexcytokine analysis of untransduced T cells or T cells transduced with19z1, P28BB, or both 48 hours post first antigen stimulation usingeither untransduced PC3 cells (Empty) or CD19⁺PSMA⁺ PC3 cells. Errorbars represent standard deviation from the mean of 2 biologicalreplicates. (B-D) depict multiplex cytokine analysis of untransduced Tcells or T cells transduced with Hz] (B), MzI (C), and Lz1 (D) anti-PSCACARs, P28BB CCRs, or both CAR+CCR is shown 48 hours post second antigenstimulation using either Empty or PSCA+PSMA+PC3 cells. (E) depictsWestern blot analysis for Bc1xL performed using cellular lysates ofuntransduced T cells or T cells transduced with 19z1, P28BB, or bothafter 24 hours post initial antigen stimulation. Total amount of Akt wasused as a loading control.

FIG. 5 depicts generation of prostate tumor cells for the expression offusion protein GFP-Firefly Luciferase (GFP/Luc) and tumor antigens.Untransduced PC3 cells (Empty) were transduced with GFP/Luc and eitherCD19, PSMA, PSCA, or a combination of two antigens using retroviralexpression constructs. Cells were purified via double purity FACS forGFP/Luc, CD19, PSMA, and/or PSCA.

FIGS. 6A-C illustrate the tumor-sensing T cell concept. (A) depicts thatTTS cells expressing an efficient CAR, become potently stimulated byA⁺113⁺ cells to facilitate immune response against A⁺ cells. CAR⁺CCR⁺cells can bind tumor antigen A⁺ cells with a CAR that supplies CD3activation signals. This can result in short-term cell lysis. CAR⁺CCR⁺cells can bind tumor antigen B⁺ cells with a CCR that supplies CD28 andCD137 signals. This signal alone is not sufficient to induce lysis orproliferation. Only when CAR⁺CCR⁺ cells bind tumor antigen A⁺B⁺ cellswith a CAR and CCR can both activation and stimulation be provided. Thisresults in robust lysis, T cell proliferation, enhanced cytokinesecretion, upregulation of BcIxL, and the ability to selectivelyeradicate tumors in vivo. However, depending on the efficacy of the CAR,these CAR⁺CCR⁺ cells can potentially recirculate to lyse cells singlepositive for antigen specific to the CAR. FIG. 6B depicts that byreducing the efficacy of the CAR, TT's cells can be functionally rescuedby CCR binding when A⁺B⁺ cells are encountered to selectively respondand eradicate A⁺B⁺ cells, while avoiding response to A+ cells. (C) showsthat by co-expressing one CAR that supplies a TCR activation signal uponbinding a tumor antigen and a second CAR that supplies stimulationsignals upon binding a different tumor antigen, T lymphocytes will onlyeradicate tumors expressing both antigens, but not tumors expressingeither antigen alone.

DETAILED DESCRIPTION OF THE INVENTION

All patents, published applications and other references mentioned inthis specification are herein incorporated by reference into the presentdisclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “activates an immunoresponsive cell” is meant induction of signaltransduction or changes in protein expression in the cell resulting ininitiation of an immune response. For example, when CD3 Chains clusterin response to ligand binding and immunoreceptor tyrosine-basedinhibition motifs (ITAMs) a signal transduction cascade is produced. Incertain embodiments, when an endogenous TCR or an exogenous CAR bindsantigen, a formation of an immunological synapse occurs that includesclustering of many molecules near the bound receptor (e.g. CD4 or CD8,CD3 / / / , etc.) This clustering of membrane bound signaling moleculesallows for ITAM motifs contained within the CD3 chains to becomephosphorylated. This phosphorylation in turn initiates a T cellactivation pathway ultimately activating transcription factors, such asNF-KB and AP-1. These transcription factors induce global geneexpression of the T cell to increase IL-2 production for proliferationand express master regulator T cell proteins in order to initiate a Tcell mediated immune response. By “stimulates an immunoresponsive cell”is meant a signal that results in a robust and sustained immuneresponse. In various embodiments, this occurs after immune cell (e.g.,T-cell) activation or concomitantly mediated through receptorsincluding, but not limited to, CD28, CD137 (4-1BB), OX40, and ICOS.Without being bound to a particular theory, receiving multiplestimulatory signals is important to mount a robust and long-term T cellmediated immune response. Without receiving these stimulatory signals, Tcells quickly become inhibited and unresponsive to antigen. While theeffects of these co-stimulatory signals vary and remain partiallyunderstood, they generally result in increasing gene expression in orderto generate long lived, proliferative, and anti-apoptotic T cells thatrobustly respond to antigen for complete and sustained eradication.

The term “antigen recognizing receptor” as used herein refers to areceptor that is capable of activating an immune cell (e.g., a T-cell)in response to antigen binding. Exemplary antigen recognizing receptorsmay be native or endogenous T cell receptors or chimeric antigenreceptors in which a tumor antigen-binding domain is fused to anintracellular signaling domain capable of activating activating animmune cell (e.g., a T-cell). In various embodiments, an antigenrecognizing receptor is selected to have low or minimal affinity oravidity for the antigen.

By “affinity” is meant a measure of the binding strength betweenantibody and a simple hapten or antigen determinant. Without being boundto theory, affinity depends on the closeness of stereochemical fitbetween antibody combining sites and antigen determinants, on the sizeof the area of contact between them, and on the distribution of chargedand hydrophobic groups. Affinity also includes the term “avidity,” whichrefers to the strength of the antigen-antibody bond after formation ofreversible complexes. Methods for calculating the affinity of anantibody for an antigen are known in the art, including use of bindingexperiments to calculate affinity. In the case of an antibody (Ab)binding to an antigen (Ag), the affinity constant is used (expressed asinverted dissociation constant).

Ab + Ag = AbAg$K_{a} = {\frac{\lbrack{AbAg}\rbrack}{\left. \lbrack{Ab}) \right\rbrack \lbrack{Ag}\rbrack} = \frac{1}{K_{a}}}$

The chemical equilibrium of antibody binding is also the ratio of theon-rate (k forward) and off-rate_((kback)) constants. Two antibodies canhave the same affinity, but one may have both a high on- and off-rateconstant, while the other may have both a low on- and off-rate constant.

$K_{a} = {\frac{\,^{k}{forward}}{\,^{k}{back}} = \frac{{on}\text{-}{rate}}{{off}\text{-}{rate}}}$

Antibody activity in functional assays (e.g., cell lysis assay) is alsoreflective of antibody affinity. In various embodiments of theinvention, the antigen recognizing receptor has low affinity. Lowaffinity includes micromolar and nanomolar affinities (e.g. 10⁻⁵, 50⁻⁶,10⁻⁶, 5×10⁻⁷, 10⁻⁷, 5×10⁻⁸, 10⁻⁸, 5×10⁻⁹, 10⁻⁹ M). Anitbody andaffinities can be phenotypically characterized and compared usingfunctional assay (e.g., cell lysis assay).

By “affinity” is meant a measure of the binding strength betweenantibody and a simple The term “chimeric co-stimulatory receptor” (CCR),as used herein refers to a specific type of chimeric antigen receptor(CAR) that mediates costimulation independently of activation. Whenexpressed on immunoresponsive cells in combination with an antigenrecognizing receptor (e.g., CAR or TCR that activates the cell), the CCRis targeted to a second antigen. In certain embodiments, the CCR has midor high affinity for its target antigen.

The term “chimeric antigen receptor” (CAR) as used herein refers to atumor antigen-binding domain that is fused to an intracellular signalingdomain capable of activating or stimulating T cells. Most commonly, theCAR's extracellular binding domain is composed of a single chainvariable fragment (scFv) derived from fusing the variable heavy andlight regions of a murine or humanized monoclonal antibody.Alternatively, scFv's may be used that are derived from Fab's (insteadof from an antibody, e.g., obtained from Fab libraries). In variousembodiments, this scFv is fused to a transmembrane domain and then tointracellular signaling domain. “First-generation” CARs include thosethat solely provide CD3 signals upon antigen binding,“Second-generation” CARs include those that provide both costimulation(e.g. CD28 or CD137) and activation (CD3). “Third-generation” CARsinclude those that provide multiple costimulation (e.g. CD28 and CD137)and activation (CD3). In CAR applications to date, the CAR is selectedto have high affinity or avidity for the antigen, which is distinct anddistinguishable from the invention described herein.

By “CD3 polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to NCBI Reference No: NP_(—)932170 or afragment thereof that has activating or stimulatory activity. Anexemplary CD3 is provided in Table 1 below. By “CD3 nucleic acidmolecule” is meant a polynucleotide encoding a CD3 polypeptide.

By “CD8 polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to NCBI Reference No: NP_(—)001759 or afragment thereof that has stimulatory activity. An exemplary CD8 isprovided in Table 1 below. By “CD8 nucleic acid molecule” is meant apolynucleotide encoding a CD8 polypeptide.

By “CD28 polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to NCBI Reference No: NP_(—)006130 or afragment thereof that has stimulatory activity. An exemplary CD28 isprovided in Table 1 below. By “CD28 nucleic acid molecule” is meant apolynucleotide encoding a CD28 polypeptide.

By “4-1 BB polypeptide” is meant a protein having at least 85, 90, 95,96, 97, 98, 99 or 100% identity to NCBI Reference No: P41273 orNP_(—)001552 or a fragment thereof that that acts as a tumor necrosisfactor (TNF) ligand. An exemplary 4-1 BB is provided in Table 1 below.By “4-1 BBL nucleic acid molecule” is meant a polynucleotide encoding a4-1BBL polypeptide.

By “CD80 polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to NCBI Reference No: NP_(—)005182 or afragment thereof that acts as an Ig superfamily ligand. An exemplaryCD80 polypeptide is provided in Table 1 below.

By “CD80 nucleic acid molecule” is meant any polynucleotide encoding aCD80 polypeptide. An exemplary CD80 nucleic acid molecule isNM_(—)005191.

By “OX4OL polypeptide” is meant a protein having at least 85, 90, 95,96, 97, 98, 99 or 100% identity to NCBI Reference No: BAB18304 orNP_(—)003317 or a fragment thereof that is a tumor necrosis factor (TNF)ligand. By “OX4OL nucleic acid molecule” is meant a polynucleotideencoding a OX4OL polypeptide.

By “19z1 polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to the sequence provided below and havingactivating activity when bound to CD19.

By “P28z polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to the sequence provided below.

By “CD19” is meant a protein having at least 85, 90, 95, 96, 97, 98, 99or 100% identity to the sequence provided below and is able to bindCD19.

By “PSMA” is meant a protein having at least 85, 90, 95, 96, 97, 98, 99or 100% identity to the sequence provided below and is able to bindPSMA.

By “P28BB” is meant a protein having at least 85, 90, 95, 96, 97, 98, 99or 100% identity to the sequence provided below and having stimulatoryactivity when bound to PSMA.

TABLE 1 SEQ ID NO. Name  1 CD3 ζ mkwkalftaa ilqaqlpite aqsfglldpkicylldgilf iygviltalf lrvkfsrsad apayqqgqnq lynelnlgrr eeydvldkrrgrdpemggkp grrknpqegl ynelqkdkma eayseigmkg errrgkghdg lyqglstatkdtydalhm qalppr  2 CD8 malpvtalll plalllhaar psqfrvspldrtwnlgetve lkcqvllsnp tsgcswlfqp rgaaasptfl lylsqnkpka aegldtqrfsgkrlgdtfvl tlsdfrrene gyyfcsalsn simyfshfvp vflpakpttt paprpptpaptiasqplslr peacrpaagg avhtrgldfa cdiyiwapla gtcgvlllsl vitlycnhrnrrrvckcprp vvksgdkpsl saryv  3 CD28 mlrlllalnl fpsiqvtgnk ilvkqspmlvaydnavnlsc kysynlfsre fraslhkgld savevcvvyg nysqqlqvys ktgfncdgklgnesvtfylq nlyvnqtdiy fckievmypp pyldneksng tiihvkgkhl cpsplfpgpskpfwvlvvvg gvlacysllv tvafiifwvr skrsrllhsd ymnmtprrpg ptrkhyqpyapprdfaayrs  4 4-1BB mgnscyniva tlllvlnfer trslqdpcsncpagtfcdnn rnqicspcpp nsfssaggqr tcdicrqckg vfrtrkecss tsnaecdctpgfhclgagcs mceqdckqgq eltkkgckdc cfgtfndqkr gicrpwtncs ldgksvlvngtkerdvvcgp spadlspgas svtppapare pghspqiisf flaltstall fllffltlrfsvvkrgrkkl lyifkqpfmr pvqttqeedg cscrfpeeee ggcel  5 CD80mghtrrqgts pskcpylnff qllvlaglsh fcsgvihvtk evkevatlsc ghnvsveelaqtriywqkek kmvltmmsgd mniwpeyknr tifditnnls ivilalrpsd egtyecvvlkyekdafkreh laevtlsvka dfptpsisdf eiptsnirrl icstsggfpe phlswlengeelnainttvs qdpetelyav sskldfnmtt nhsfmcliky ghlrvnqtfn wnttkqehfpdnllpswait lisvngifvi ccltycfapr crerrrnerl rresvrpv  6 OX40Lmervqpleen vgnaarprfe rnklllvasv iqglglllcf tyiclhfsal qvshrypriqsikvqfteyk kekgfiltsq kedeimkvqn nsviincdgf ylislkgyfs qevnislhyqkdeeplfqlk kvrsvnslmv asltykdkvy lnvttdntsl ddfhvnggel ilihqnpgef cvl  719z1 MALPVTALLLPLALLLHAEVKLQQSGAELVRP GSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSST AYMQLSGLTSEDSAVYECARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELT QSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGT DFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAPTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRVKFSRSAEPPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR  8P28z MALPVTALLLPLALLLHAEVQLQQSGPELVKP GTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSST AYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMS TSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIT NVQSEDLADYFCQQYNSYPLTFGAGTMLDLKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCP SPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFTIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR  9 CD19 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKF KGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSG GGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR 10 PSMA MMALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSL EWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYNELRSLTSEDSAVYYCAAGWNFDYWGQGT TVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQS PKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK R 11 P28BBMALPVTALLLPLALLLHAEVQLQQSGPELVKP GTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSST AYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMS TSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIT NVQSEDLADYFCQQYNSYPLTFGAGTMLDLKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCP SPLFPGPSKPFWVINVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 1001.1 g/m1denatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 p.g/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42.degree C. in 15 mM NaCl, 1.5 mM trisodiumcitrate, and 0.1% SDS. In another embodiment, wash steps will occur at68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Hybridization techniques are well known to those skilled in the art andare described, for example, in Benton and Davis (Science 196:180, 1977);Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975);Ausubel et al. (Current Protocols in Molecular Biology, WileyInterscience, New York, 2001); Berger and Kimmel (Guide to MolecularCloning Techniques, 1987, Academic Press, New York); and Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

By “analog” is meant a structurally related polypeptide or nucleic acidmolecule having the function of a reference polypeptide or nucleic acidmolecule.

The term “ligand” as used herein refers to a molecule that binds to areceptor. In particular, the ligand binds a receptor on another cell,allowing for cell-to-cell recognition.

The term “constitutive expression” as used herein refers to expressionunder all physiological conditions.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasia or pathogen infection of cell.

By “effective amount” is meant an amount sufficient to arrest,ameliorate, or inhibit the continued proliferation, growth, ormetastasis (e.g., invasion, or migration) of a neoplasia.

By “enforcing tolerance” is meant preventing the activity ofself-reactive cells or immunoresponsive cells that target transplantedorgans or tissues.

By “exogenous” is meant a nucleic acid molecule or polypeptide that isnot endogenously present in the cell, or not present at a levelsufficient to achieve the functional effects obtained whenover-expressed. The term “exogenous” would therefore encompass anyrecombinant nucleic acid molecule or polypeptide expressed in a cell,such as foreign, heterologous, and over-expressed nucleic acid moleculesand polypeptides.

By a “heterologous nucleic acid molecule or polypeptide” is meant anucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptidethat is not normally present in a cell or sample obtained from a cell.This nucleic acid may be from another organism, or it may be, forexample, an mRNA molecule that is not normally expressed in a cell orsample.

By “immunoresponsive cell” is meant a cell that functions in an immuneresponse or a progenitor, or progeny thereof.

By “isolated cell” is meant a cell that is separated from the molecularand/or cellular components that naturally accompany the cell.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that canbe subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

The term “tumor antigen-binding domain” as used herein refers to adomain capable of specifically binding a particular antigenicdeterminant or set of antigenic determinants present on a tumor.

By “modulate” is meant to alter positively or negatively. Exemplarymodulations include a 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100% change.

By “neoplasia” is meant a disease characterized by the pathologicalproliferation of a cell or tissue and its subsequent migration to orinvasion of other issues or organs. Neoplasia growth is typicallyuncontrolled and progressive, and occurs under conditions that would notelicit, or would cause cessation of, multiplication of normal cells.Neoplasias can affect a variety of cell types, tissues, or organs,including but not limited to an organ selected from the group consistingof bladder, bone, brain, breast, cartilage, glia, esophagus, fallopiantube, gallbladder, heart, intestines, kidney, liver, lung, lymph node,nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin,spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,urogenital tract, ureter, urethra, uterus, and vagina, or a tissue orcell type thereof. Neoplasias include cancers, such as sarcomas,carcinomas, or plasmacytomas (malignant tumor of the plasma cells).Illustrative neoplasms for which the invention can be used include, butare not limited to leukemias (e.g., acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemi a, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, ligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma). In oneembodiment, screening methods of the invention identify compositionsthat are useful for treating breast or lung cancer.

By “receptor” is meant a polypeptide, or portion thereof, present on acell membrane that selectively binds one or more ligands.

By “recognize” is meant selectively binds a target. A T cell thatrecognizes a virus typically expresses a receptor that binds an antigenexpressed by the virus.

By “pathogen” is meant a virus, bacteria, fungi, parasite or protozoacapable of causing disease. Exemplary viruses include, but are notlimited to, Retroviridae (e.g. human immunodeficiency viruses, such asHIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III;and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bungs viruses,phleboviruses and Nairo viruses); Arena Viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andlridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.Hepatitis C); Norwalk and related viruses, and astroviruses).

Exemplary bacteria include, but are not limited to, Pasteurella,Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, andSalmonella species. Specific examples of infectious bacteria include butare not limited to, Helicobacter pyloris, Borelia burgdoiferi,Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M.avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyo genes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pat hogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Rickettsia, and Actinomyces israelli.

By “specifically binds” is meant a polypeptide or fragment thereof thatrecognizes and binds a polypeptide of interest, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

The term “tumor antigen” as used herein refers to any polypeptideexpressed by a tumor that is capable of inducing an immune response.

By “virus antigen” is meant a polypeptide expressed by a virus that iscapable of inducing an immune response.

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the disease course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Therapeutic effects of treatment include,without limitation, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, preventing metastases,decreasing the rate of disease progression, amelioration or palliationof the disease state, and remission or improved prognosis. By preventingprogression of a disease or disorder, a treatment can preventdeterioration due to a disorder in an affected or diagnosed subject or asubject suspected of having the disorder, but also a treatment mayprevent the onset of the disorder or a symptom of the disorder in asubject at risk for the disorder or suspected of having the disorder.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a human.

The term “immunocompromised” as used herein refers to a subject who hasan immunodeficiency. The subject is very vulnerable to opportunisticinfections, infections caused by organisms that usually do not causedisease in a person with a healthy immune system, but can affect peoplewith a poorly functioning or suppressed immune system.

Other aspects of the invention are described in the following disclosureand are within the ambit of the invention.

The present invention generally provides cells, including geneticallymodified immunoresponsive cells (e.g., T cells, Natural Killer (NK)cells, cytotoxic T lymphocytes (CTL) cells) expressing at least acombination of an antigen-recognizing receptor (e.g., TCR or CAR) and achimeric co-stimulating receptor (CCR), and methods of use therefore forthe treatment of neoplasia and other pathologies where an increase in anantigen-specific immune response is desired. The invention is based, atleast in part, on the discovery that the simultaneous engagement of twoantigens co-expressed by a tumor cell by an antigen-recognizing receptorand chimeric co-stimulating receptor is useful for activating andstimulating an immunoreactive cell without systemic effects. Inparticular, the reactivity against tissues expressing either antigenalone is preferably minimal, inducing T cell activation in the presenceof both antigens but not either one alone. T cell activation is mediatedby a TCR or a CAR targeted to an antigen (e.g., CD19 or prostate stemcell antigen, PSCA). Costimulation is independently mediated by a“chimeric costimulatory receptor” (CCR),^(12,13) which is targeted to asecond antigen (e.g., prostate-specific membrane antigen, PSMA). Such anapproach resulted in augmented reactivity against dual-antigen positive(DP) tumors, but failed to avert enhanced reactivity against singleantigen positive (SP) tumors. It was found that tumor sensing T cellscould be made to differentiate DP tumors from SP tumors by attenuating Tcell activation to a level where T cell activation is by itselfineffective, but functionally rescued at the tumor site by a CCR engagedby an independent, co-expressed antigen. This approach providesimmunogenicity within the tumor microenvironment for tumor eradicationwhile not affecting SP cells that are normal or non-neoplastic andrepresents a significant advance over conventional adoptive T celltherapy.

Furthermore, this approach is not limited to the treatment ofneoplasias, but is amenable to a wide range of applications where anincrease in an antigen-specific immune response is desired, suchapplications include not only the treatment of neoplasias, but also forthe enhancement of an immune response against a pathogen infection or aninfectious disease and to reinforce immune tolerance in regulatory Tcells in the context of autoimmunity or allogeneic transplantation.

Hematopoietic Cell Lineages

Mammalian hematopoietic (blood) cells provide a diverse range ofphysiologic activities. Hematopoietic cells are divided into lymphoid,myeloid and erythroid lineages. The lymphoid lineage, comprising B, Tand natural killer (NK) cells, provides for the production ofantibodies, regulation of the cellular immune system, detection offoreign agents in the blood, detection of cells foreign to the host, andthe like. The term “T cells” as used herein refers to lymphocytes thatmature in the thymus and are chiefly responsible for cell-mediatedimmunity. T cells are involved in the adaptive immune system. The term“natural killer (NK) cells” as used herein refers to lymphocytes thatare part of cell-mediated immunity and act during the innate immuneresponse. They do not require prior activation in order to perform theircytotoxic effect on target cells. Cytotoxic T cells (CTL or killer Tcells) are a subset of T lymphocytes capable of inducing the death ofinfected somatic or tumor cells.

Cells for Use in the Methods of the Invention

The present invention provides cells expressing a combination of anantigen-recognizing receptor that activates an immunoresponsive cell(e.g., TCR, CAR) and a chimeric co-stimulating receptor (CCR), andmethods of using such cells for the treatment of a disease that requiresan enhanced immune response. In one approach, tumor antigen-specific Tcells, NK cells, CTL cells or other immunoresponsive cells are used asshuttles for the selective enrichment of one or more co-stimulatoryligands for the treatment or prevention of neoplasia. For example, a Tcell expressing a chimeric antigen receptor 19z1 that recognizes CD19 isco-expressed in a T cell that expresses a chimeric co-stimulatoryreceptor P28BB that recognizes and binds Prostate Specific MembraneAntigen (PSMA). Such cells are administered to a human subject in needthereof for the treatment or prevention of prostate cancer. In anotherapproach, viral antigen-specific T cells, NK cells, CTL cells can beused for the treatment of viral diseases. For example, a chimericco-stimulatory antigen receptor that recognizes a first CMV antigen anda chimeric antigen receptor that recognizes and binds a second CMVantigen are co-expressed in cytotoxic T lymphocytes for the treatment ofCMV.

Tumor Antigen-Specific T Lymphocytes (and NK Cells)

Types of tumor antigen-specific human lymphocytes that can be used inthe methods of the invention include, without limitation, peripheraldonor lymphocytes genetically modified to express chimeric antigenreceptors (CARs) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45),peripheral donor lymphocytes genetically modified to express afull-length tumor antigen-recognizing T cell receptor complex comprisingthe a and p heterodimer (Morgan, R. A., et al. 2006 Science314:126-129), lymphocyte cultures derived from tumor infiltratinglymphocytes (TILs) in tumor biopsies (Panelli, M. C., et al. 2000 JImmunol 164:495-504; Panelli, M. C., et al. 2000 J Immunol164:4382-4392), and selectively in vitro-expanded antigen-specificperipheral bloodleukocytes employing artificial antigen-presenting cells(AAPCs) or pulsed dendritic cells (Dupont, J., et al. 2005 Cancer Res65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102:2498-2505). TheT cells may be autologous, allogeneic, or derived in vitro fromengineered progenitor or stem cells.

Any suitable tumor antigen (antigenic peptide) is suitable for use inthe tumor-related embodiments described herein. Sources of antigeninclude, but are not limited to cancer proteins. The antigen can beexpressed as a peptide or as an intact protein or portion thereof. Theintact protein or a portion thereof can be native or mutagenized.Suitable antigens include prostate specific membrane antigen (PSMA) andprostate stem cell antigen (PCSA).

Viral Antigen-Specific T Lymphocytes (and NK Cells)

Suitable antigens for use in the treatment of pathogen infection orother infectious disease, for example, in an immunocompromised subjectinclude, without limitation, viral antigens present in Cytomegalovirus(CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), andinfluenza virus.

The unpurified source of CTLs may be any known in the art, such as thebone marrow, fetal, neonate or adult or other hematopoietic cell source,e.g., fetal liver, peripheral blood or umbilical cord blood. Varioustechniques can be employed to separate the cells. For instance, negativeselection methods can remove non-CTLs initially. mAbs are particularlyuseful for identifying markers associated with particular cell lineagesand/or stages of differentiation for both positive and negativeselections.

A large proportion of terminally differentiated cells can be initiallyremoved by a relatively crude separation. For example, magnetic beadseparations can be used initially to remove large numbers of irrelevantcells. Preferably, at least about 80%, usually at least 70% of the totalhematopoietic cells will be removed prior to cell isolation.

Procedures for separation include, but are not limited to, densitygradient centrifugation; resetting; coupling to particles that modifycell density; magnetic separation with antibody-coated magnetic beads;affinity chromatography; cytotoxic agents joined to or used inconjunction with a mAb, including, but not limited to, complement andcytotoxins; and panning with antibody attached to a solid matrix, e.g.plate, chip, elutriation or any other convenient technique.

Techniques for separation and analysis include, but are not limited to,flow cytometry, which can have varying degrees of sophistication, e.g.,a plurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels.

The cells can be selected against dead cells, by employing dyesassociated with dead cells such as propidium iodide (PI). Preferably,the cells are collected in a medium comprising 2% fetal calf serum (FCS)or 0.2% bovine serum albumin (BSA) or any other suitable, preferablysterile, isotonic medium.

Accordingly, the invention generally provides an immunoresponsive cell,such as a virus specific or tumor specific T cell comprising a receptorthat binds a first antigen and activates the immunresponsive cell and areceptor that binds a second antigen and stimulates the immunresponsivecell.

Vectors

Genetic modification of immunoresponsive cells (e.g., T cells, CTLcells, NK cells) can be accomplished by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct.Preferably, a retroviral vector (either gamma-retroviral or lentiviral)is employed for the introduction of the DNA construct into the cell. Forexample, a polynucleotide encoding a receptor that binds an antigen(e.g., a tumor antigen, or a variant, or a fragment thereof), can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retroviral long terminal repeat, or from apromoter specific for a target cell type of interest. Non-viral vectorsmay be used as well.

For initial genetic modification of the cells to provide tumor or viralantigen-specific cells, a retroviral vector is generally employed fortransduction, however any other suitable viral vector or non-viraldelivery system can be used. For subsequent genetic modification of thecells to provide cells comprising an antigen presenting complexcomprising at least two co-stimulatory ligands, retroviral gene transfer(transduction) likewise proves effective. Combinations of retroviralvector and an appropriate packaging line are also suitable, where thecapsid proteins will be functional for infecting human cells. Variousamphotropic virus-producing cell lines are known, including, but notlimited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437);PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP(Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464).Non-amphotropic particles are suitable too, e.g., particles pseudotypedwith VSVG, RD114 or GALV envelope and any other known in the art.

Possible methods of transduction also include direct co-culture of thecells with producer cells, e.g., by the method of Bregni, et al. (1992)Blood 80:1418-1422, or culturing with viral supematant alone orconcentrated vector stocks with or without appropriate growth factorsand polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat.22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

Other transducing viral vectors can be used to express a co-stimulatoryligand of the invention in an immunoresponsive cell. Preferably, thechosen vector exhibits high efficiency of infection and stableintegration and expression (see, e.g., Cayouette et al., Human GeneTherapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844,1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini etal., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad.Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be usedinclude, for example, adenoviral, lentiviral, and adeno-associated viralvectors, vaccinia virus, a bovine papilloma virus, or a herpes virus,such as Epstein-Barr Virus (also see, for example, the vectors ofMiller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281,1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al.,Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research andMolecular Biology 36:311-322, 1987; Anderson, Science 226:401-409,1984;Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; andJohnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularlywell developed and have been used in clinical settings (Rosenberg etal., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No.5,399,346).

Non-viral approaches can also be employed for the expression of aprotein in cell. For example, a nucleic acid molecule can be introducedinto a cell by administering the nucleic acid in the presence oflipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am.J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247:1465, 1990).

Other non-viral means for gene transfer include transfection in vitrousing calcium phosphate, DEAE dextran, electroporation, and protoplastfusion. Liposomes can also be potentially beneficial for delivery of DNAinto a cell. Transplantation of normal genes into the affected tissuesof a subject can also be accomplished by transferring a normal nucleicacid into a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue or are injectedsystemically. Recombinant receptors can also be derived or obtainedusing transposases or targeted nucleases (e.g. Zinc finger nucleases,meganucleases, or TALE nucleases). Transient expression may be obtainedby RNA electroporation. cDNA expression for use in polynucleotidetherapy methods can be directed from any suitable promoter (e.g., thehuman cytomegalovirus (CMV), simian virus 40 (SV40), or metallothioneinpromoters), and regulated by any appropriate mammalian regulatoryelement or intron (e.g. the elongation factor 1cenhancer/promoter/intron structure). For example, if desired, enhancersknown to preferentially direct gene expression in specific cell typescan be used to direct the expression of a nucleic acid. The enhancersused can include, without limitation, those that are characterized astissue- or cell-specific enhancers. Alternatively, if a genomic clone isused as a therapeutic construct, regulation can be mediated by thecognate regulatory sequences or, if desired, by regulatory sequencesderived from a heterologous source, including any of the promoters orregulatory elements described above.

The resulting cells can then be grown under conditions similar to thosefor unmodified cells, whereby the modified cells can be expanded andused for a variety of purposes.

Also included in the invention are 19z1, CD19, CD8, CD3, dsRed, P28BB,PSMA, CD28, 4-1 BB, GFP polypeptides or fragments thereof that aremodified in ways that enhance their anti-neoplastic activity whenexpressed in an immunoresponsive cell. The invention provides methodsfor optimizing an amino acid sequence or nucleic acid sequence byproducing an alteration in the sequence. Such alterations may includecertain mutations, deletions, insertions, or post-translationalmodifications. The invention further includes analogs of anynaturally-occurring polypeptide of the invention. Analogs can differfrom a naturally-occurring polypeptide of the invention by amino acidsequence differences, by post-translational modifications, or by both.Analogs of the invention will generally exhibit at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with all or partof a naturally-occurring amino, acid sequence of the invention. Thelength of sequence comparison is at least 5, 10, 15 or 20 amino acidresidues, preferably at least 25, 50, or 75 amino acid residues, andmore preferably more than 100 amino acid residues. Again, in anexemplary approach to determining the degree of identity, a BLASTprogram may be used, with a probability score between e³ and e⁻¹″indicating a closely related sequence. Modifications include in vivo andin vitro chemical derivatization of polypeptides, e.g., acetylation,carboxylation, phosphorylation, or glycosylation; such modifications mayoccur during polypeptide synthesis or processing or following treatmentwith isolated modifying enzymes. Analogs can also differ from thenaturally-occurring polypeptides of the invention by alterations inprimary sequence. These include genetic variants, both natural andinduced (for example, resulting from random mutagenesis by irradiationor exposure to ethanemethylsulfate or by site-specific mutagenesis asdescribed in Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra).Also included are cyclized peptides, molecules, and analogs whichcontain residues other than L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g.,—or—amino acids.

In addition to full-length polypeptides, the invention also providesfragments of any one of the polypeptides or peptide domains of theinvention. As used herein, the term “a fragment” means at least 5, 10,13, or 15 amino acids. In other embodiments a fragment is at least 20contiguous amino acids, at least 30 contiguous amino acids, or at least50 contiguous amino acids, and in other embodiments at least 60 to 80,100, 200, 300 or more contiguous amino acids. Fragments of the inventioncan be generated by methods known to those skilled in the art or mayresult from normal protein processing (e.g., removal of amino acids fromthe nascent polypeptide that are not required for biological activity orremoval of amino acids by alternative mRNA splicing or alternativeprotein processing events).

Non-protein analogs have a chemical structure designed to mimic thefunctional activity of a protein of the invention. Such analogs areadministered according to methods of the invention. Such analogs mayexceed the physiological activity of the original polypeptide. Methodsof analog design are well known in the art, and synthesis of analogs canbe carried out according to such methods by modifying the chemicalstructures such that the resultant analogs increase the anti-neoplasticactivity of the original polypeptide when expressed in animmunoresponsive cell. These chemical modifications include, but are notlimited to, substituting alternative R groups and varying the degree ofsaturation at specific carbon atoms of a reference polypeptide.Preferably, the protein analogs are relatively resistant to in vivodegradation, resulting in a more prolonged therapeutic effect uponadministration. Assays for measuring functional activity include, butare not limited to, those described in the Examples below.

Co-Stimulatory Ligands

The interaction with at least one co-stimulatory ligand provides anon-antigenspecific signal important for full activation of an immunecell (e.g., T cell). Co-stimulatory ligands include, without limitation,tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12,IL-15 or IL21), and immunoglobulin (Ig) superfamily ligands.

Tumor necrosis factor (TNF) is a cytokine involved in systemicinflammation and stimulates the acute phase reaction. Its primary roleis in the regulation of immune cells. Tumor necrosis factor (TNF)ligands share a number of common features. The majority of the ligandsare synthesized as type II transmembrane proteins(extracellularC-terminus) containing a short cytoplasmic segment and arelatively long extracellular region. TNF ligands include, withoutlimitation, nerve growth factor (NGF), CD4OL (CD4OL)/CD154,CD137L/4-1BBL, tumor necrosis factor alpha (TNFa), CD134L/OX4OL/CD252,CD27L/CD70, Fas ligand (FasL), CD3OL/CD153, tumor necrosis factor beta(TNF(3)/lymphotoxin-alpha (LTa), lymphotoxin-beta (ur(3), CD257/Bcell-activating factor (BAFF)/Blys/THANK/Ta11-1, glucocorticoid-inducedTNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand(TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a largegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. These proteinsshare structural features with immunoglobulins—they possess animmunoglobulin domain (fold). Immunoglobulin superfamily ligandsinclude, without limitation, CD80 and CD86, both ligands for CD28.

Compositions comprising genetically modified immunoresponsive cells ofthe invention (e.g., T cells, NK cells, CTL cells, or their progenitors)can be provided systemically or directly to a subject for the treatmentof a neoplasia, pathogen infection, or infectious disease. In oneembodiment, cells of the invention are directly injected into an organof interest (e.g., an organ affected by a neoplasia). Alternatively,compositions comprising genetically modified immunoresponsive cells areprovided indirectly to the organ of interest, for example, byadministration into the circulatory system (e.g., the tumorvasculature). Expansion and differentiation agents can be provided priorto, during or after administration of the cells to increase productionof T cells, NK cells, or CTL cells in vitro or in vivo.

The modified cells can be administered in any physiologically acceptablevehicle, normally intravascularly, although they may also be introducedinto bone or other convenient site where the cells may find anappropriate site for regeneration and differentiation (e.g., thymus).Usually, at least 1×10⁵ cells will be administered, eventually reaching1×10¹⁰, or more. Genetically modified immunoresponsive cells of theinvention can comprise a purified population of cells. Those skilled inthe art can readily determine the percentage of genetically modifiedimmunoresponsive cells in a population using various well-known methods,such as fluorescence activated cell sorting (FACS). Preferable ranges ofpurity in populations comprising genetically modified immunoresponsivecells are about 50 to about 55%, about 55 to about 60%, and about 65 toabout 70%. More preferably the purity is about 70 to about 75%, about 75to about 80%, about 80 to about 85%; and still more preferably thepurity is about 85 to about 90%, about 90 to about 95%, and about 95 toabout 100%. Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage). Thecells can be introduced by injection, catheter, or the like. If desired,factors can also be included, including, but not limited to,interleukins, e.g. IL-2, IL-3, IL-6, and IL-11, as well as the otherinterleukins, the colony stimulating factors, such as G-, M- and GM-CSF,interferons, e.g. .gamma.-interferon and erythropoietin.

Compositions of the invention include pharmaceutical compositionscomprising genetically modified immunoresponsive cells or theirprogenitors and a pharmaceutically acceptable carrier. Administrationcan be autologous or heterologous. For example, immunoresponsive cells,or progenitors can be obtained from one subject, and administered to thesame subject or a different, compatible subject. Peripheral bloodderived immunoresponsive cells of the invention or their progeny (e.g.,in vivo, ex vivo or in vitro derived) can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, or parenteraladministration. When administering a therapeutic composition of thepresent invention (e.g., a pharmaceutical composition containing agenetically modified immunoresponsive cell), it will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion).

Formulations

Compositions of the invention comprising genetically modifiedimmunoresponsive cells can be conveniently provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may be buffered to aselected pH. Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating thegenetically modified immunoresponsive cells utilized in practicing thepresent invention in the required amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionsmay be in admixture with a suitable carrier, diluent, or excipient suchas sterile water, physiological saline, glucose, dextrose, or the like.The compositions can also be lyophilized. The compositions can containauxiliary substances such as wetting, dispersing, or emulsifying agents(e.g., methylcellulose), pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the genetically modified immunoresponsive cells or theirprogenitors.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount that will achieve the selectedviscosity. Obviously, the choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form).

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the genetically modifiedimmunoresponsive cells as described in the present invention. This willpresent no problem to those skilled in chemical and pharmaceuticalprinciples, or problems can be readily avoided by reference to standardtexts or by simple experiments (not involving undue experimentation),from this disclosure and the documents cited herein. One considerationconcerning the therapeutic use of genetically modified immunoresponsivecells of the invention is the quantity of cells necessary to achieve anoptimal effect. The quantity of cells to be administered will vary forthe subject being treated. In a one embodiment, between 10⁴ to 10¹⁰,between 10⁵ to 10⁹, or between 10⁶ and 10⁸ genetically modifiedimmunoresponsive cells of the invention are administered to a humansubject. More effective cells may be administered in even smallernumbers. In some embodiments, at least about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,and 5×10⁸ genetically modified immunoresponsive cells of the inventionare administered to a human subject. The precise determination of whatwould be considered an effective dose may be based on factors individualto each subject, including their size, age, sex, weight, and conditionof the particular subject. Dosages can be readily ascertained by thoseskilled in the art from this disclosure and the knowledge in the art.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active cell(s) and/or agent(s)) are present in an amountof 0.001 to 50% (weight) solution in phosphate buffered saline, and theactive ingredient is present in the order of micrograms to milligrams,such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1wt %, still more preferably about 0.0001 to about 0.05 wt % or about0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, andstill more preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD50 in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Methods of Treatment

Provided herein are methods for treating neoplasia in a subject. Alsocontemplated herein are methods for treating a pathogen infection orother infectious disease in a subject, such as an immunocompromisedhuman subject. The methods comprise administering a T cell, NK cell, orCTL cell of the invention in an amount effective to achieve the desiredeffect, be it palliation of an existing condition or prevention ofrecurrence. For treatment, the amount administered is an amounteffective in producing the desired effect. An effective amount can beprovided in one or a series of administrations. An effective amount canbe provided in a bolus or by continuous perfusion.

An “effective amount” (or, “therapeutically effective amount”) is anamount sufficient to effect a beneficial or desired clinical result upontreatment. An effective amount can be administered to a subject in oneor more doses. In terms of treatment, an effective amount is an amountthat is sufficient to palliate, ameliorate, stabilize, reverse or slowthe progression of the disease, or otherwise reduce the pathologicalconsequences of the disease. The effective amount is generallydetermined by the physician on a case-by-case basis and is within theskill of one in the art. Several factors are typically taken intoaccount when determining an appropriate dosage to achieve an effectiveamount. These factors include age, sex and weight of the subject, thecondition being treated, the severity of the condition and the form andeffective concentration of the antigen-binding fragment administered.

For adoptive immunotherapy using antigen-specific T cells, cell doses inthe range of 10⁶-10¹⁰ (e.g., 10⁹) are typically infused. Uponadministration of the genetically modified cells into the host andsubsequent differentiation, T cells are induced that are specificallydirected against the specific antigen. “Induction” of T cells caninclude inactivation of antigen-specific T cells such as by deletion oranergy. Inactivation is particularly useful to establish or reestablishtolerance such as in autoimmune disorders. The modified cells can beadministered by any method known in the art including, but not limitedto, intravenous, subcutaneous, intranodal, intratumoral, intrathecal,intrapleural, intraperitoneal and directly to the thymus.

The invention provides methods for increasing an immune response in asubject in need thereof. In one embodiment, the invention providesmethods for treating or preventing a neoplasia in a subject. Theinvention provides therapies that are particularly useful for thetreatment of subjects having prostate cancer, or metastatic prostatecancer that is not amenable to conventional therapeutic interventions.Suitablehuman subjects for therapy typically comprise two treatmentgroups that can be distinguished by clinical criteria. Subjects with“advanced disease” or “high tumor burden” are those who bear aclinically measurable tumor. A clinically measurable tumor is one thatcan be detected on the basis of tumor mass (e.g., by palpation, CATscan, sonogram, mammogram or X-ray; positive biochemical orhistopathologic markers on their own are insufficient to identify thispopulation). A pharmaceutical composition embodied in this invention isadministered to these subjects to elicit an anti-tumor response, withthe objective of palliating their condition. Ideally, reduction in tumormass occurs as a result, but any clinical improvement constitutes abenefit. Clinical improvement includes decreased risk or rate ofprogression or reduction in pathological consequences of the tumor.

A second group of suitable subjects is known in the art as the “adjuvantgroup.” These are individuals who have had a history of neoplasia, buthave been responsive to another mode of therapy. The prior therapy canhave included, but is not restricted to, surgical resection,radiotherapy, and traditional chemotherapy. As a result, theseindividuals have no clinically measurable tumor. However, they aresuspected of being at risk for progression of the disease, either nearthe original tumor site, or by metastases. This group can be furthersubdivided into high-risk and low-risk individuals. The subdivision ismade on the basis of features observed before or after the initialtreatment. These features are known in the clinical arts, and aresuitably defined for each different neoplasia. Features typical ofhigh-risk subgroups are those in which the tumor has invaded neighboringtissues, or who show involvement of lymph nodes.

Another group have a genetic predisposition to neoplasia but have notyet evidenced clinical signs of neoplasia. For instance, women testingpositive for a genetic mutation associated with breast cancer, but stillof childbearing age, can wish to receive one or more of theantigen-binding fragments described herein in treatment prophylacticallyto prevent the occurrence of neoplasia until it is suitable to performpreventive surgery.

Human neoplasia subjects having any of the following neoplasias:glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, softtissue sarcoma, and various carcinomas (including prostate and smallcell lung cancer) are especially appropriate subjects. Suitablecarcinomas further include any known in the field of oncology,including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma,liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitiveneural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma,pancreatic ductal adenocarcinoma, small and large cell lungadenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamouscell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, andliver metastases thereof, lymphangiosarcoma,lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma,mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basalcell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceousgland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,testicular tumor, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma,Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumorssuch as ductal and lobular adenocarcinoma, squamous and adenocarcinomasof the uterine cervix, uterine and ovarian epithelial carcinomas,prostatic adenocarcinomas, transitional squamous cell carcinoma of thebladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma,acute and chronic leukemias, malignant melanoma, soft tissue sarcomasand leiomyosarcomas.

The subjects can have an advanced form of disease, in which case thetreatment objective can include mitigation or reversal of diseaseprogression, and/or amelioration of side effects. The subjects can havea history of the condition, for which they have already been treated, inwhich case the therapeutic objective will typically include a decreaseor delay in the risk of recurrence.

Accordingly, the invention provides a method of treating or preventing aneoplasia in a subject, the method comprising administering an effectiveamount of an immunoresponsive cell comprising a receptor that binds atumor antigen and activates the immunoresponsive cell (e.g., TCR, CAR)and a vector encoding a receptor that binds another tumor antigen andstimulates the immunoresponsive cell. In one embodiment, the neoplasiais selected from the group consisting of prostate cancer, breast cancer,blood cancers (e.g. leukemias, lymphomas, and myelomas), ovarian cancer,bladder cancer, brain cancer, colon cancer, intestinal cancer, livercancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer,stomach cancer, glioblastoma, and throat cancer. In another embodiment,the tumor antigen is one or more of carbonic anhydrase IX (CAIX),carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30,CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, anantigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surfaceantigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40(EGP-40), epithelial cell adhesion molecule (EpCAM), receptortyrosine-protein kinases erb-B2,3,4, folate-binding protein (FBP), fetalacetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2),Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2),human telomerase reverse transcriptase (hTERT), Interleukin-13 receptorsubunit alpha-2 (IL-13Ra2), ic-light chain, kinase insert domainreceptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM),melanoma antigen family A, 1 (MAGE-A1), Mucin 1 (MUC1), Mesothelin(MSLN), NKG2D ligands, cancer-testis antigen NY-ES0-1, oncofetal antigen(h5T4), prostate stem cell antigen (PSCA), prostate-specific membraneantigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascularendothelial growth factor R2 (VEGF-R2), or Wilms tumor protein (WT-1).

As a consequence of surface expression of a receptor that binds a tumorantigen and activates the immunoresponsive cell (e.g., TCR, CAR) and avector encoding a receptor that binds another tumor antigen andstimulates the immunoresponsive cell (e.g. CCR), adoptively transferredhuman T or NK cells are endowed with augmented and selective cytolyticactivity at the tumor site. Furthermore, subsequent to theirlocalization to tumor or viral infection and their proliferation,co-stimulatory ligandexpressing T cells turn the tumor or viralinfection site into a highly conductive environment for a wide range ofimmune cells involved in the physiological anti-tumor or antiviralresponse (tumor infiltrating lymphocytes, NK-, NKT-cells, dendriticcells, and macrophages).

In other embodiments, the invention provides methods for treatingsubjects with a pathogen infection (e.g., viral infection, bacterialinfection, fungal infection, parasite infection, or protozoalinfection). The invention is particularly useful for enhancing an immuneresponse in an immunocompromised subject. Exemplary viral infectionssusceptible to treatment using a method of the invention include, butare not limited to, Cytomegalovirus (CMV), Epstein Barr Virus (EBV),Human Immunodeficiency Virus (HIV), and influenza virus infections.

Accordingly, the invention provides a method of treating or preventing apathogen infection in a subject, the method comprising administering aneffective amount of an immunoresponsive cell as described herein.

Kits

The invention provides kits for the treatment or prevention of aneoplasia, pathogen infection, immune disorder or allogeneic transplant.In one embodiment, the kit includes a therapeutic or prophylacticcomposition containing an effective amount of an immunoresponsive cellcomprising an activating antigen receptor and a co-stimulatory antigenreceptor in unit dosage form. In particular embodiments, the cellsfurther comprise a co-stimulatory ligand. In some embodiments, the kitcomprises a sterile container which contains a therapeutic orprophylactic vaccine; such containers can be boxes, ampules, bottles,vials, tubes, bags, pouches, blister-packs, or other suitable containerforms known in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingmedicaments.

If desired the immunoresponsive cell is provided together withinstructions for administering the cell to a subject having or at riskof developing a neoplasia, pathogen infection, immune disorder orallogeneic transplant. The instructions will generally includeinformation about the use of the composition for the treatment orprevention of neoplasia, pathogen infection, immune disorder orallogeneic transplant. In other embodiments, the instructions include atleast one of the following: description of the therapeutic agent; dosageschedule and administration for treatment or prevention of a neoplasia,pathogen infection, immune disorder or allogeneic transplant or symptomsthereof; precautions; warnings; indications; counter-indications;overdosage information; adverse reactions; animal pharmacology; clinicalstudies; and/or references. The instructions may be printed directly onthe container (when present), or as a label applied to the container, oras a separate sheet, pamphlet, card, or folder supplied in or with thecontainer.

EXAMPLES

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

Example 1 T Cells Co-Expressing a Chimeric Antigen Receptor (CAR) and aChimeric Co-Stimulating Receptor (CCR) Eradicated Established Tumors

The invention provides “tumor-sensing T cells” that simultaneouslyengage two antigens co-expressed by a tumor cell. Importantly, it hasbeen found that the reactivity against tissues expressing either antigenalone should be negligible, only unleashing T cell activation in thepresence of both antigens but not either one alone. The invention is atleast based in part on the discoveries that in combination provideselective T cell immunoreactivity, and, thus, make this approachclinically relevant. The first is to assign T cell activation to oneantigen (e.g., CD19 or prostate stem cell antigen, PSCA), which may bemediated by a T cell receptor (TCR) or a chimeric antigen receptor(CAR). Costimulation is independently mediated by a “chimericcostimulatory receptor” (CCR),^(12,13) which is targeted to a secondantigen (e.g., prostate-specific membrane antigen, PSMA). This approachresulted in increased immunoreactivity against dual antigen positive(DP) tumors, but failed to avert enhanced immunoreactivity againstsingle antigen positive (SP) tumors. The second principle important fortumor sensing T cells to differentiate DP tumors from SP tumors, is todiminish T cell activation to a level where it is by itself ineffective,but functionally rescued at the tumor site by a CCR engaged by anindependent, co-expressed antigen. As CARs and CCRs recognize cellsurface antigens rather than HLA-peptide complexes, T cells engineeredin this manner are directly targeted to the tumor and will not becostimulated by interacting with cells cross-presenting the targetedantigens. As demonstrated herein, this approach resulted in selectivetumor eradication in multiple tumor-bearing mice.

To demonstrate that both T cell activation and costimulation signals canbe supplied in vivo using two distinct antigen-specific receptors, thecombination of a CAR, providing a CD3 activation signal upon recognitionof the B cell marker CD19¹⁴ and a CCR specific for PSMA^(12,15) wasevaluated. Because of the synergy between CD28 and 4-1BB^(16,17),including tandem cytoplasmic domains¹⁸⁻²¹, 4-1BB cytoplasmic domain wasadded to the PSMA CCR P28¹⁵ as described²′ (FIG. 1A). Primary humanperipheral blood T cells were transduced with the 19z1 and/or P28BBreceptors and showed readily detectable expression of both receptors,with transduction efficiencies in the range of 4570% (FIG. 1B). Fourgroups of T cells were analyzed in all subsequent studies, comprisinganti-CD19 CAR (19z1), anti-PSMA CCR (P28BB), both anti-CD19 CAR andanti-PSMA CCR in combination (19z1+P28BB), and a mock-transduced controlgroup (mock) (FIG. 1C). The in vitro cytotoxic and proliferativeresponse upon exposure to CD19 and/or PSMA showed that cytotoxicitydirected against CD19 was, as expected, imparted by 19z1 and unalteredin the presence of PSMA.

A quantitative comparison of the T cell groups, normalized to thefraction of 19z1-transduced T cells for the 19z1 and 19z1+P28BB groupsand the P28BBtransduced fraction in the P28BB group showed that 19z1 and19z1+P28BB T cells specifically lysed 40-47% CD19⁺ targets at the 50:1E:T ratio while the P28BB-transduced T cells failed to lyse PSMA⁺targets (FIG. 2A). However, upon repeated exposure to these antigens inthe absence of exogenous cytokine, only the 19z1+P28BB T cells exhibitedrobust proliferation with a 58-fold expansion for 31 days whenco-cultured on artificial antigen presenting cells (AAPCs) thatexpressed both antigens. By comparison, 19z1 or P28BB T cells onlydisplayed modest expansion over the first 14 days, as did the 19z1+P28BBT cells on CD19⁺PSMA⁺ APCs (FIG. 2B). Further evidence of stronger Tcell activation in the presence of both antigens was provided by thequantitative assessment of cytokine production and the induction of theantiapoptotic molecule BcIxL in 19z1+P28BB T cells, which weredistinctly greater in the presence of CD19⁺PSMA⁺ APCs than in thepresence of either antigen alone (FIGS. 4A, 4E).

Initially, the in vivo ability of these dual-receptor expressing T cellsto eradicate established systemic human prostate tumors inimmunocompromised NOD/SCID-yC KO (NSG) mice bearing dual-positive(CD19+PSMA+) tumor cells was tested. The NSG mice were systemicallyengrafted with 2.0×10⁶ firefly-luciferase expressing PC3 tumor cellsthat expressed both CD19 and PSMA (FIG. 5) and treated 19 days laterwith a single intravenous infusion of 1.0×10⁶ 19 z1, 19z1+P28BB, P28BBor control T cells. Thirty-five days later, mice that received P28BB Tcells or control T cells were sacrificed due to tumor burden. Incontrast, mice treated with 19z1 T cells had a marked reduction of tumorburden. Strikingly, mice treated with 19z1+P28BB T cells hadundetectable tumor burden (FIG. 2C). Over 70 days of post-infusionmonitoring, the CD19⁺ tumors eventually relapsed in mice that received19z1 T cells, while complete remission persisted in all mice thatreceived 19z1+P28BB T cells (FIG. 2C). This result strongly indicatedthat tumor eradication had been achieved.

These findings however posed a concern because of the potential baseline eradication of CD19⁺PSMA⁻ tumors by 19z1+P28BB T cells. There was alikelihood that T cell immunoreactivity would be undesirably enhanced inrecipients bearing dual-positive CD19⁺PSMA⁺ tumors due to recirculationof T cells. To test this hypothesis, mice were subcutaneously infusedwith CD19⁺PSMA⁺ tumors into the left flanks, CD19⁺ PSMA⁺ tumors into theright flanks, and CD19⁺PSMA⁺ tumors into their backs. One week later,mice were administered one of 19z1, P28BB, or 19z1+P28BB T cells(1.0×10⁶ cells) intravenously. Mice that received P28BB T cells hadprogression of all three tumors and needed to be sacrificed within 35days (FIG. 2D). In mice treated with 19z1 T cells, the CD19⁺PSMA⁺ andCD19⁺PSMA⁺ tumors underwent a substantial reduction compared to theirprogression in recipients of P28BB T cells, before eventuallyprogressing. Consistent with prior results, mice treated with 19z1+P28BBT cells showed complete eradication of CD19⁺PSMA⁺ tumors. However, ashypothesized, rejection of CD19⁺PSMA⁺ tumors was also substantiallyenhanced and superior to that observed in recipients of 19z1 T cells(FIG. 2D, lower panels). Thus, a split signal approach targeting twoantigens failed to restrict T cell reactivity and to protect singleantigen tumors.

To address the problem of single antigen reactivity, it was proposedthat T cell activation would have to be minimized, almost to the pointof extinction, only to be rescued at the site of dual antigen expressingby an adequate CCR engagement. Thus, CARs with diminished activity weresought. For these experiments, a clinically relevant combination ofantigens targeting PSCA and PSMA were used. Three PSCA-specific scFvswere evaluated with different binding affinities for PSCA (FIG. 3A).While the HzI scFv efficiently lysed tumor cells down to the picogramrange, the LzI scFv required 1,000-10,000 fold more antibody to achievesimilar efficiency of specific lysis. These scFvs were used to derivethree CD3-based CARs with different activities in cytotoxicity assays(FIG. 3B). Two of the CARs, HzI and MzI, directed moderate lyticactivity against PSCA⁺ targets (20% specific lysis at the 50:1 E:Tratio). In contrast the third CAR, LzI, only reached 10%, qualifying itas a inefficient antigen receptor. This hierarchy was further confirmedin cytokine release assays, which showed enhanced cytokine secretion by19z1+P28BB T cells (FIG. 4A) and Hz1+P28BB T cells (FIG. 4B) compared tocells with either receptor alone. This enhancement was less in MzI+P28BBT cells (FIG. 4C) and even further decreased in Lz1+P28BB T cells (FIG.4D).

In order to evaluate the therapeutic efficacy and targeting profile ofPSCA+PSMA− reactive T cells, the anti-tumor activity of these T cellswas tested in animals bearing PSCA+PSMA− and/or PSCA+PSMA+ tumors.First, to test the ability of MzI+P28BB and Lz1+P28BB T cells toeradicate PSCA+PSMA+ cells selectively, mice were inoculatedintravenously with 2×106 FFLuc-expressing PC3 cells positive for PSMA,PSCA, or both (FIG. 5). Fourteen days later, one set of mice received1×106 MzI+P28BB CAR+T cells infused intravenously, and another set ofreceived 1×106 Lz1+P28BB CARP T cells. Mice bearing PSCA+PSMA tumorcells that were treated with the more efficient MzI+P28BB T cellsexhibited greater tumor regression than mice treated with Lz1+PBB Tcells (FIG. 3C). Similar to the CD19 experiment (FIG. 2C), these tumorseventually relapsed and progressed. However, in mice bearing PSCA+PSMA+tumor cells MzI+P28BB T cells induced robust and long-term tumoreradication. Consistent with the lesser potency of LzI+P28BB T cells,tumor eradication in mice bearing PSCA+PSMA+ tumor cells treated withLz1+P28BB T cells was slower but nonetheless equally successful,resulting in strong tumor eradication and long-term survival of alltreated mice (FIG. 3C). Tumor eradication was not enhanced in controlmice bearing either PSCAVSMA− or PSCA-PSMA+ tumors (FIG. 3C). A morestringent evaluation of background activity against PSCAVSMA− tumors wastested in the context of animals also bearing PSCA+PSMA+ and PSCAPSMA+tumors. Lz1+P28BB T cells mediated eradication of PSCA+PSMA+ tumorswithout increasing eradication of PSCA+PSMA tumors (FIG. 3E), which wasnot different from that induced by LzI T cells.

Thus, these results demonstrate the feasibility of decreasing T cellactivation to the point of averting immune reactivity against tissuesexpressing one targeted antigen and rescuing T cell activation at thetumor site where two antigens are co-expressed, without running the riskof igniting reactivity against the single antigen-expressing tissues. Indoing so, the results demonstrate proof-of-principle for achieving twocomplementary outcomes that determine specificity and safety: 1) theability to create targeting specificity in the absence of a uniquetarget antigen through combinatorial antigen recognition; and 2) theprotection of cells expressing only one of the antigens by titratingactivation and costimulatory signals, so as to practically confineactivation to sites of target antigen coexpression.

Targeted T cell therapies have the potential to provide curativetreatments but their applicability is limited by the paucity ofvalidated tumor-specific targets. Extra-tumoral expression resultsindeed in “on-target, off-tumor” effects that 2-4″ may be sometimestolerable but are eventually lethall 11. The method described hereinprovides improved targeting by supplying titrated activation andcostimulation signals through combinatorial antigen recognition (FIGS.6A-6C).

In physiological antigen presentation,²² T cells are primed in lymphnodes by receiving activating and costimulatory signals and migrate toperipheral sites, where effector functions of the T cells are not asdependent on costimulation. Similarly, T cells engaged through anantigen receptor and a CCR may recirculate to other peripheral sites anddisplay heightened cytolytic activity against tissues expressing onlyone of the targeted antigens (FIG. 6A). Therefore, the present strategywas developed to address this problem of a potential systemic effect inorder to spare cells singly positive for the antigen, includingnon-tumor cells (FIG. 6B). In a tri-tumor mouse model (PSCA⁺, PSMA⁺, andPSCA⁺PSMA⁺), eradication of PSCAVSMA⁺ was accomplished, while sparingthe PSCA⁺PSMA″ and PSCA⁻PSMA⁺ tumors (FIG. 3E).

As shown by the present studies, this selectivity for DP tumors can beachieved by reducing the efficacy of the CAR, which creates cells thatare less cytotoxic (FIG. 3B) and that have reduced levels of cytokinesecretion (FIGS. 4A-4D). While the levels of both TH I and TH2 cytokinesare relatively high for both 19z1 and HzI CARs, using less efficientCARs MzI and LzI resulted in reducing these levels. The enhancement ofcytokine levels in LzI+P28BB T cells compared to LzI T cells was minimalexcept for IL-2 and IL-13. While IL-2 induces proliferation and canpromote either a TH 1 or TH2 response²³, IL-13 is associated with a TH2response specific to 4-I BB/CD1 37 signaling.^(24,25)

PSCA and PSMA are promising targets for the treatment of metastaticprostate cancer^(26,27), although neither is absolutelyprostate-specific. In human subjects, PSCA expression is found inprostate cancer and within the renal pelvis, ureter, urinary bladder,and urethra.²⁸ Expression of PSMA strongly correlates with primaryprostate cancer, metastases, as well as in astrocytes type II, thekidney proximal tubule and the intestinal brush border.²⁹ Dual PSCA/PSMAtargeting is thus expected to increase prostate cancer targeting andreduce reactivity against these normal tissues. It is appreciated thatthis principle can be extended to other tumor types which express a pairof antigens, especially those that confer true tumor-specificity. Forexample, HER2, MUC1, CD44, CD49f, and/or EpCAM could be used in thismanner to treat breast cancer.^(3,1) Likewise, mesothelin, folatereceptor-a, CD44, and/or CD133 could be used to treat ovariancancer.^(32, 33) The targeting of tumor initiating cells or cancer stemcells, for which unique target antigens/structures have not yet beenclearly identified34.3s would be particularly attractive using thisapproach.

An important aspect of this approach is to constrain and nearly abolishT cell activation in response to a single antigen. CARs with lowaffinity or low avidity that only provide a poor activation signal werefound to be useful for achieving this effect. Alternatively, anendogenous TCR with low affinity or low avidity may be used incombination with a CCR to provide antigen-specific costimulation.Altogether, the results indicate the advantages of restricting theactivity of engineered T cells, reconciling potency with safety throughcombinatorial antigen recognition by tumor-sensing T cells.

Gammaretroviral Vector Construction and Viral Production

The gammaretroviral vector SFG-19z1 has been extensively described.¹⁴This backbone construct was used to exchange scFvs to generate SFG-HzI,SFG-MzI, and SFG-LzI by directional cloning utilizing a NcoI sitelocated 5′ of the scFv and a Nod site located 3′ of the scFv. Togenerate SFG-P28BB, the fused CD28 and CD137 domains were PCR amplifiedfrom SFG-P28BBz1 and ligated 3′ of the PSMA scFv using a 5′ NcoI siteand a 3′ BamHI site to include a stop codon 3′ of the BB domain, whilethe CD3 domain was removed.²¹ Bicistronic gene expression for CARs to becoexpressed with dsRED and CCRs to be coexpressed with hrGFP wasachieved by using an Internal Ribosomal Entry Site as previouslydescribed. Vectors were used to transiently transfect cell lines togenerate stable viral producing lines as previously described.

Generation of Anti-PSCA scFvs

Three novel PSCA specific scFvs, termed HzI, MzI, and LzI were generatedbyamplifying the variable heavy (VH) and variable light (VL) domainsconferring PSCA antigen specificity of non-overlapping epitopes usingdegenerate primers from hybridomas as previously described.³⁶ These VHand V_(L) domains were fused together using a linker and were used toreplace the CD19 scFv in the SFG-19z1 backbone using 5′SphI to 3′NotIsites.

Isolation, Retroviral Transduction, and Culture of Primary Human T Cells

Peripheral blood leukocytes were isolated using Ficoll gradients andtransduced as previously described. Briefly, after 48-hour activationwith 2 &g/mL phytohemagglutinin, cells were transduced twice viaspinoculation for 1 hour on retronectin coated plates over the next 48hours and 20 U/mL of IL-2 was added. After allowing 3 days for vectorexpression, transduction efficiencies were determined via flow cytometryand bulk unsorted cells were used for various assays or adoptivetransfers.

Generation of Antigen Expressing Tumor Cell Lines

The PC3 human prostate tumor line was obtained from ATCC andretrovirally¹⁶ transduced in order to generate PC3-GFP/Luc, which wassubsequently used to create 10 PC3-CD19, PC3-PSMA, PC3-CD19-PSMA,PC3-PSCA, and PC3-PSCA-PSMA via retroviral transduction.

CTL Chromium Release Killing Assays

Target cells expressing desired antigen were labeled with ⁵¹Cr andco-cultured with T cells at decreasing effector:target ratio's. After 4hours of culture, supernatant was removed and used to measureradioactivity released from chromium. Specific lysis was determined bysubtracting background radioactivity of target cells not cultured with Tcells and dividing by the radioactivity measured from target cellscompletely lysed by using 0.2% Triton X-100.

Long-Term T Cell Proliferation Assays

Tumor cells expressing desired antigen were irradiated with 30 Gy priorto co-culture with 1.0×10⁶ T cells at a 5:1 effector:target ratio. Tcells were counted weekly using an Invitrogen Countess cell counter andthen re-stimulated with irradiated tumor cells. No exogenous cytokineswere added to these co-cultures.

Generation of Tumor Models in Mice

PC3 tumor cells were infused into NOD/SCID-IL2Ry mice obtained fromeither Jackson Laboratories or from in-house breeding under the protocol04-10-024 approved by the MSKCC Institutional Animal Care and UseCommittee. For systemic tumor experiments, 2.0×10⁶ tumor infused intomice with 1.0×10⁶ chimeric receptor positive T cells infused 14 dayslater. For subcutaneous tumor experiments, 1.0×10⁶ tumor cells wereinjected per tumor site, established for 7 days upon which 1.0×10⁶chimeric positive T cells were infused IV.

Quantification of Tumor Burden

For systemic tumor experiments, bioilluminescent imaging (BLI) was usedto quantitatively measure tumor burden by correlating the amount oftumor burden to luminescence using an IVIS 100 system (Caliper LifeSciences) as previously described. For subcutaneous tumors, caliperswere used to measure tumor size. Tumor volume was calculated bymultiplying the length, width, and height of each tumor.

Bispecific Antibody Mediated Tumor Lysis

Bispecific antibodies containing a PSCA specific scFv fused to a CD3specific scFv were added at various amounts to untransduced T cellsco-cultured with PSCA⁺ PC3 at a 20:1 ratio, respectively in standard 4hr chromium release assay assays.

Flow Cytometry

Cells were analyzed using an LSRII flow cytometer or sorted using aFACSAria cell sorter (BD Biosciences) as previously described.¹⁶Detection of chimeric receptor at the cell surface could be achieveddirectly by using AF647 conjugated goat-anti-mouse antibody(Invitrogen). Antibodies for CD4-PE-Cy7, CD8-Pacific Blue, and CD19-APCwere obtained from Invitrogen while PSCA antibodies were purified fromhybridoma supernatants and PSMA antibodies were obtained from MBLInterantional.

Cytokine Analysis

Supernatants harvested 48 hours after the second tumor stimulation fromlong-term T cell proliferation experiments and were used for cytokineanalysis by using a custom multiplex system HCYTMAG-60K (Millipore) andanalyzed using a Luminex 100 instrument (Luminex) as previouslydescribed.²¹

Western Blot Analysis

Cells were harvested 24 hours after initial tumor stimulation fromlong-term T cell proliferation experiments to be used for western blotanalysis of BcI_(x)L expression. Western blots were performed aspreviously described²¹ using BcI_(x)L and Akt primary antibodies (CellSignaling Technology).

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

This application may be related to U.S. patent application Ser. No.12/593,751, which is the U.S. national phase application, pursuant to 35U.S.C. §371, of International Patent Application No.: PCT/US20081004251,filed Mar. 8, 2010, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/921,144, filed Mar. 30, 2007, the disclosures ofwhich are hereby incorporated herein in their entireties by reference.

REFERENCES

-   1. Robbins, P. F. et al. Tumor regression in patients with    metastatic synovial cell sarcoma and melanoma using genetically    engineered lymphocytes reactive with NY-ESO-1. Journal of clinical    oncology: official journal of the American Society of Clinical    Oncology 29, 917-924 (2011).-   2. Kalos, M. et al. T cells with chimeric antigen receptors have    potent antitumor effects and can establish memory in patients with    advanced leukemia. Sci Transl Med 3, 95ra73 (2011).-   3. Brentjens, R. J. et al. Safety and persistence of adoptively    transferred autologous CD19-ta rgeted T cells in patients with    relapsed or chemotherapy refractory B-cell leukemias. Blood 118,    4817-4828 (2011).-   4. Kochenderfer, J. N. et al. B-cell depletion and remissions of    malignancy along with cytokine-associated toxicity in a clinical    trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.    Blood 119, 2709-2720 (2012).-   5. Sadelain, M., Riviere, I. & Brentjens, R. Targeting tumours with    genetically enhanced T lymphocytes. Nat Rev Cancer 3, 35-45 (2003).-   6. Ho, W. Y., Blattman, J. N., Dossett, M. L., Yee, C. &    Greenberg, P. D. Adoptive immunotherapy: engineering T cell    responses as biologic weapons for tumor mass destruction. Cancer    cell 3, 431-437 (2003).-   7. Rosenberg, S. A., Restifo, N. P., Yang, J. C., Morgan, R. A. &    Dudley, M. E. Adoptive cell transfer: a clinical path to effective    cancer immunotherapy. Nature reviews. Cancer 8, 299-308 (2008).-   8. Sadelain, M., Brentjens, R. & Riviere, I. The promise and    potential pitfalls of chimeric antigen receptors. Curr Opin Immunol    21, 215-223 (2009).-   9. Jorritsma, A., Schotte, R., Coccoris, M., de Witte, M. A. &    Schumacher, T. N. Prospects and limitations of T cell receptor gene    therapy. Current gene therapy 11, 276-287 (2011).-   10. Johnson, L. A. et al. Gene therapy with human and mouse T, cell    receptors mediates cancer regression and targets normal tissues    expressing cognate antigen. Blood 114, 535-546 (2009).-   11. Morgan, R. A. et al. Case report of a serious adverse event    following the administration of T cells transduced with a chimeric    antigen receptor recognizing ERBB2. Molecular therapy: the journal    of the American Society of Gene Therapy 18, 843-851 (2010).-   12. Krause, A. et al. Antigen-dependent CD28 signaling selectively    enhances survival and proliferation in genetically modified    activated human primary T lymphocytes. J Exp Med 188, 619-626    (1998).-   13. Wilkie, S. et al. Dual Targeting of ErbB2 and MUC1 in Breast    Cancer Using Chimeric Antigen Receptors Engineered to Provide    Complementary Signaling. Journal of clinical immunology (2012).-   14. Brentjens, R. J. et al. Eradication of systemic B-cell tumors by    genetically targeted human T lymphocytes co-stimulated by CD80 and    interleukin-15. Nature medicine 9, 279-286 (2003).-   15. Maher, J., Brentjens, R. J., Gunset, G., Riviere, I. &    Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation    directed by a single chimeric TCRzeta/CD28 receptor. Nat Bio    technol. 20, 70-75 (2002).-   16. Stephan, M. T. et al. T cell-encoded CD80 and 4-1BBL induce    auto- and transcostimulation, resulting in potent tumor rejection.    Nature medicine 13, 1440-1449 (2007).-   17. Watts, T. H. TNF/TNFR family members in costimulation of T cell    responses. Annual review of immunology 23, 23-68 (2005).-   18. Wang, J. et al. Optimizing adoptive polyclonal T cell    immunotherapy of lymphomas, using a chimeric T cell receptor    possessing CD28 and CD137 costimulatory domains. Human gene therapy    18, 712-725 (2007).-   19. Carpenito, C. et al. Control of large, established tumor    xenografts with genetically retargeted human T cells containing CD28    and CD137 domains. Proceedings of the National Academy of Sciences    of the United States of America 106, 3360-3365 (2009).-   20. Tammana, S. et al. 4-1BB and CD28 signaling plays a synergistic    role in redirecting umbilical cord blood T cells against B-cell    malignancies. Hum Gene Ther 21, 75-86 (2010).-   21 Zhong, X. S., Matsushita, M., Plotkin, J., Riviere, I. &    Sadelain, M. Chimeric antigen receptors combining 4-1BB and CD28    signaling domains augment PI3kinase/AKT/Bc1-X L activation and C    D8+T cell-mediated tumor eradication. Molecular therapy: the journal    of the American Society of Gene Therapy 18, 413-420 (2010).-   22. Schwartz, R. H. T cell anergy. Annual review of immunology 21,    305-334 (2003).-   23. Liao, W., Lin, J. X & Leonard, W. J. IL-2 family cytokines: new    insights into the complex roles of IL-2 as a broad regulator of T    helper cell differentiation. Current opinion in immunology 23,    598-604 (2011).-   24. Nam, K. O., Shin, S M. & Lee, H. W. Cross-linking of 4-IBB    up-regulates IL-13 expression in CD8(+) T lymphocytes. Cytokine 33,    87-94 (2006).-   25. Shin, S M et al. 4-IBB triggers IL-13 production from T cells to    limit the polarized, Thl-mediated inflammation. Journal of leukocyte    biology 81, 1455-1465 (2007).-   26. Saeki, N., Gu, J., Yoshida, T. & Wu, X. Prostate stem cell    antigen: a Jekyll and Hyde molecule? Clinical cancer research: an    official journal of the American Association for Cancer Research 16,    3533-3538 (2010).-   27. Olson, W. C., Heston, W. D. & Rajasekaran, A. K. Clinical trials    of cancer therapies targeting prostate-specific membrane antigen.    Reviews on recent clinical trials 2, 182-190 30 (2007).-   28. Lam, J. S. et al. Prostate stem cell antigen is overexpressed in    prostate cancer metastases. Clinical cancer research: an official    journal of the American Association for Cancer Research 11,    2591-2596 (2005).-   29. Silver, D. A., Pellicer, I., Fair, W. R., Heston, W. D. &    Cordon-Cardo, C. Prostate-specific membrane antigen expression in    normal and malignant human tissues. Clinical cancer research: an    official journal of the American Association for Cancer Research 3,    81-85 (1997).-   30. Liu, J. C. et al. Seventeen-gene signature from enriched    Her2/Neu mammary tumor-initiating cells predicts clinical outcome    for human HER2+:ERalpha-breast cancer. Proceedings of the National    Academy of Sciences of the United States of America 109, 58325837    (2012).-   31. Meyer, M. J. et al. CD44posCD49fhiCD133/2hi defines    xenograft-initiating cells in estrogen receptor-negative breast    cancer. Cancer research 70, 4624-4633 (2010).-   32. Strauss, R. et al. Analysis of epithelial and mesenchymal    markers in ovarian cancer reveals phenotypic heterogeneity and    plasticity. PloS one 6, el 6186 (2011).-   33. Shihle, M. & Davidson, B. Pathogenesis of ovarian cancer: clues    from selected overexpressed genes. Future Oncol 5, 1641-1657 (2009).-   34. Nguyen, L. V., Vanner, R., Dirks, P. & Eaves, C. J. Cancer stem    cells: an evolving concept. Nature reviews. Cancer 12, 133-143    (2012).-   35. Magee, J. A., Piskounova, E. & Morrison, S. J. Cancer stem    cells: impact, heterogeneity, and uncertainty. Cancer cell 21,    283-296 (2012).-   36. Orlandi, R., Gussow, D. H., Jones, P. T. & Winter, G. Cloning    immunoglobulin variable domains for expression by the polymerase    chain reaction. Proceedings of the National Academy of Sciences of    the United States of America 86, 3833-3837 (1989).

What is claimed is:
 1. An immunoresponsive cell comprising: a. anantigen recognizing receptor that binds a first antigen with lowaffinity, wherein binding of the receptor to the first antigen activatesthe immunoresponsive cell, and b. a chimeric co-stimulating receptor(CCR) that binds a second antigen and stimulates the immunoresponsivecell.
 2. The immunoresponsive cell of any one of claims 1, wherein thecell is selected from the group consisting of a T cell, a Natural Killer(NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a humanembryonic stem cell, and a pluripotent stem cell from which lymphoidcells may be differentiated.
 3. The immunoresponsive cell of claim 1 or2, wherein the antigen is a tumor or pathogen antigen.
 4. Theimmunoresponsive cell of any of claims 1-3, wherein said antigenrecognizing receptor is a T cell receptor (TCR) or chimeric antigenreceptor (CAR).
 5. The immunoresponsive cell of any one of claims 1-4,wherein said antigen recognizing receptor is exogenous or endogenous. 6.The immunoresponsive cell of any one of claims 1-5, wherein said antigenrecognizing receptor is recombinantly expressed.
 7. The immunoresponsivecell of any one of claims 1-6, wherein the antigen recognizing receptoris expressed from a vector.
 8. The immunoresponsive cell of any one ofclaims 1-7, wherein the chimeric co-stimulating receptor (CCR) isexpressed from a vector.
 9. The immunoresponsive cell of any one ofclaims 1-8, wherein said immunoresponsive cell is autologous.
 10. Theimmunoresponsive cell of any one of claims 1-9, wherein said first andsecond antigens are selected from the group consisting of CAIX, CEA,CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44,CD49f, CD56, CD74, CD133, CD138, a cytomegalovirus (CMV) infected cellantigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, Fetal acetylcholinereceptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, x-lightchain, KDR, LeY, LI cell adhesion molecule, MAGE-AI, MUC1, Mesothelin,NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1,TAG-72, VEGF-R2, or WT-1.
 11. The immunoresponsive cell of any one ofclaims 1-9, wherein said first and second antigens are distinct antigensselected from the group consisting of CD133, a cytomegalovirus (CMV)infected cell antigen, erb-B2, KDR Mesothelin, NKG2D ligands, NY-ES0-1,oncofetal antigen (h5T4), PSCA, PSMA, CD19, VEGF-R2, and WT-1.
 12. Theimmunoresponsive cell of any one of claims 1-9, wherein said first andsecond antigens are selected from the group consisting of HER2, MUC1,CD44, CD49f, EpCAM, CEA, CD133, a cytomegalovirus (CMV) infected cellantigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, KDR, Mesothelin, NKG2Dligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, VEGF-R2, orWT-1.
 13. The immunoresponsive cell of any one of claims 1-11, whereinsaid first and second antigens are CD10 and CD19.
 14. Theimmunoresponsive cell of any one of claims 1-11, wherein said first andsecond antigens are CD56 and CD138.
 15. The immunoresponsive cell of anyone of claims 1-11, wherein said first and second antigens are distinctantigens selected from the group consisting of mesothelin, folatereceptor-a, CD44, and CD133.
 16. The immunoresponsive cell of any one ofclaims 1-15, wherein the intracellular signaling domain of said antigenrecognizing receptor is the CD3-chain signaling domain.
 17. Theimmunoresponsive cell of any one of claims 1-16, wherein theintracellular signaling domain of the chimeric co-stimulating receptor(CCR) is a CD97, CD1 la-CD18, CD2, ICOS, CD27, CD154, CD5, OX40, 4-1BBor CD28 signaling domain.
 18. The immunoresponsive cell of any one ofclaims 1-17, wherein the cell expresses an antigen receptor that is 19z1or Pz1.
 19. A method of inducing tumor cell death in a tumor, the methodcomprising contacting said tumor cell with an immunoresponsive cellcomprising an antigen recognizing receptor that binds a first tumorantigen with low affinity, wherein the binding activates theimmunoresponsive cell, and a chimeric co-stimulating receptor (CCR) thatbinds a second tumor antigen and stimulates the immunoresponsive cell,thereby inducing tumor cell death in said tumor.
 20. The method of claim19, wherein the method reduces the number of tumor cells.
 21. The methodof claim 19, wherein the method reduces tumor size.
 22. The method ofclaim 19, wherein the method eradicates the tumor.
 23. A method oftreating or preventing a neoplasia in a subject, the method comprisingadministering an effective amount of an immunoresponsive cell comprisingan antigen recognizing receptor that binds a first antigen with lowaffinity, wherein the binding activates the immunoresponsive cell, and achimeric co-stimulating receptor (CCR) that binds a second antigen andstimulates the immunoresponsive cell, thereby treating or preventing aneoplasia in the subject.
 24. The method of claim 23, wherein theneoplasia is selected from the group consisting of prostate cancer,breast cancer, B cell leukemia, multiple myeloma, and ovarian cancer.25. The method of claim 23, wherein the neoplasia is breast cancer andthe first and second tumor antigens are distinct antigens selected fromthe group consisting of HER2, MUC1, CD44, CD49f, EpCAM, CEA, CD133, acytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM,erb-B2,3,4, FBP, KDR, Mesothelin, NKG2D ligands, NY-ES0-1, oncofetalantigen (h5T4), PSCA, PSMA, VEGF-R2, or WT-1.
 26. The method of claim23, wherein the neoplasia is B cell leukemia and the first and secondtumor antigens are CD10 and CD19.
 27. The method of claim 24, whereinthe neoplasia is multiple myeloma and the first and second tumorantigens are CD56 and CD138.
 28. The method of claim 24, wherein theneoplasia is ovarian cancer and the first and second tumor antigens aredistinct antigens selected from the group consisting of mesothelin,folate receptor-a, CD44, and CD133.
 29. The method of any one of claims23-28, wherein said immunoresponsive cell is selected as having anantigen recognizing receptor with low affinity.
 30. The method of anyone of claims 23-28, wherein said antigen recognizing receptor isselected for expression in the cell as having low affinity.
 31. Themethod of any one of claims 23-28, wherein said antigen recognizingreceptor is a T cell receptor (TCR) or chimeric antigen receptor (CAR).32. The method of any one of claims 23-28, wherein said antigenrecognizing receptor is exogenous or endogenous.
 33. The method of anyone of claims 23-32, wherein the antigen recognizing receptor isexpressed from a vector.
 34. The method of any one of claims 23-32,wherein the chimeric co-stimulating receptor (CCR) is expressed from avector.
 35. The method of any one of claims 23-34, wherein the cell isselected from the group consisting of a T cell, a Natural Killer (NK)cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a humanembryonic stem cell, and a pluripotent stem cell from which lymphoidcells may be differentiated.
 36. The method of any one of claims 23-35,wherein the method reduces or eradicates the tumor burden in thesubject.
 37. A method of inducing tumor cell death, the methodcomprising administering an effective amount of an immunoresponsive cellcomprising an antigen recognizing receptor that binds a first antigenwith low affinity, wherein the binding activates the immunoresponsivecell, and a chimeric co-stimulating receptor (CCR) that binds a secondantigen and stimulates the immunoresponsive cell, thereby inducing tumorcell death in the subject.
 38. A method for producing anantigen-specific immunoresponsive cell, the method comprisingintroducing into the immunoresponsive cell a nucleic acid sequence thatencodes a chimeric co-stimulating receptor (CCR), wherein the chimericco-stimulating receptor comprises an antigen-binding domain coupled toan intracellular signaling domain that stimulates an immunoresponsivecell, wherein the immunoresponsive cell comprises an antigen recognizingreceptor that binds a first antigen with low affinity, wherein thebinding activates the immunoresponsive cell.
 39. The method of claim 38,the method further comprising introducing a second nucleic acid sequencethat encodes a chimeric antigen receptor, wherein the chimeric antigenreceptor comprises a second antigen-binding domain coupled to a secondintracellular signaling domain that activates an immunoresponsive cell.40. The method of any one of claims 38-39, wherein the tumor antigensare distinct antigens selected from the list consisting of CAIX, CEA,CD5, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44,CD49f, CD56, CD74, CD133, CD138, a cytomegalovirus (CMV) infected cellantigen, EGP-2, EGP-40, EpCAM, erbB2,3,4, FBP, Fetal acetylcholinereceptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, x-lightchain, KDR, LeY, LI cell adhesion molecule, MAGE-AI, MUC1, Mesothelin,NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1,TAG-72, VEGF-R2, or WT-1.
 41. The method of any one of claims 38-40,wherein said antigen recognizing receptor is a T cell receptor (TCR) orchimeric antigen receptor (CAR).
 42. The method of any one of claims38-41, wherein said antigen recognizing receptor is exogenous orendogenous.
 43. The method of any one of claims 38-42, wherein saidantigen recognizing receptor is recombinantly expressed.
 44. The methodof any one of claims 38-43, wherein the antigen recognizing receptor isexpressed from a vector.
 45. The method of any one of claims 38-44,wherein the chimeric co-stimulating receptor (CCR) is expressed from avector.
 46. The method of any one of claims 38-45, wherein the cell isselected from the group consisting of a T cell, a Natural Killer (NK)cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a humanembryonic stem cell, and a pluripotent stem cell from which lymphoidcells may be differentiated.
 47. The method of any one of claims 38-46,wherein the intracellular signaling domain of said antigen recognizingreceptor is the CD3-chain signaling domain.
 48. The method of any one ofclaims 38-47, wherein the intracellular signaling domain of the chimericco-stimulating receptor (CCR) is a CD97, CD11a-CD18, CD2, ICOS, CD27,CD154, CD5, OX40, 4-1 BB or CD28 signaling domain.
 49. A method oftreating prostate cancer in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a T cell comprising an antigen recognizing receptor that bindsPSCA or CD19 with low affinity, wherein the binding activates theimmunoresponsive cell, and a chimeric co-stimulating receptor (CCR) thatbinds PSMA and stimulates the immunoresponsive cell, thereby treatingprostate cancer in the subject.
 50. A pharmaceutical compositioncomprising an immunoresponsive cell of any one of claims 1-18 and apharmaceutically acceptable excipient.
 53. An immunoresponsive cell ofany of claims 1-18 for use in the treatment of a neoplasia or tumordisease.
 54. A T cell comprising an antigen recognizing receptor thatbinds PSCA or CD19 with low affinity, wherein the binding activates theimmunoresponsive cell, and a chimeric co-stimulating receptor (CCR) thatbinds PSMA and stimulates the immunoresponsive cell for use in thetreatment of prostate cancer.
 55. A kit comprising an immunoresponsivecell comprising an antigen recognizing receptor that binds a firstantigen and activates the immunoresponsive cell, and a chimericco-stimulating receptor (OCR) that binds a second viral antigen andstimulates the immunoresponsive cell.